1. Field of the Invention
The present invention relates to assays, and in particular to, protease assays using site-specifically labeled fluorescent full-length protein substrates or an independently folded domain of a protein as substrates.
2. Description of Related Art
Despite natural substrates of proteases in living cells being full-length proteins, most proteases assay methods used in biological research and drug discovery employ short peptides as substrates. Peptides are small fragments of a protein (i.e., its sequence is derived from proteins). Although a peptide shares the similar or identical amino acid sequence with the corresponding segment of its parent protein, it lacks many key structural and functional features since it does not have the tertiary folding of the protein. As a result, there are serious drawbacks in using peptide-based assays for screenings, leads optimization and structure-activity relation studies to discover drugs that target proteases.
Further, the sequence of a peptide substrate is designed to be similar to that around the cleavage site of the substrate protein to give a degree of specificity. In known assays, the protein is labeled by a fluorescence dye and a quencher conjugated at opposite ends of such peptide. However, when the peptide is cleaved by a protease into two fragments, the dye and quencher become decoupled resulting in an increase of fluorescence intensity. These types of known assays are widely adopted and used as standard assay methods for almost all proteases for compound screen and structure-activity relationship driving in drug discovery, as well as kinetic and mechanism studies in biological research. However, since a short linear peptide does not have the specific conformation of its parent protein, such assays suffer serious drawbacks.
One of the drawbacks of assays using short linear peptides as the substrate is that often interaction between a protease and a substrate protein are not limited to interactions between the active sites of the enzyme and the cleavage site of the protein. Other parts of proteins are also involved in the interaction, and the overall conformation of the substrate protein may be critical to the reaction specificity. These types of interactions do not exist for a peptide substrate.
Another drawback of using short linear peptides in assays is that the binding pockets on the surface of a substrate protein are completely lost on a peptide. As such, compounds that may bind to the substrate protein may not be able to be identified on a peptide. Additionally, the pH dependency and other reaction conditions of a peptide substrate can be very different from those of the natural protein substrate.
The above problems associated with using short linear peptides in assays may be resolved by using a natural protein substrate. However, due to the technical difficulties in handling and processing natural protein substrates they are rarely used in biochemical protease assays and methods. One of the drawbacks in using natural protein substrates for protease assays is that the reaction often needs to be stopped, and the product must be separated by electrophoresis and/or chromatograph methods. These steps are not only cumbersome and time consuming, they generate a low throughput low and the results lack quantitative accuracy.
In view of the foregoing, a need exists in the art for improved methods, systems and apparatus for performing protease assays using full-length protein substrates.